This proposal is designed to examine the disruption of key metabolic events within cells that maintain arachidonic acid (AA) homeostasis as a means of impeding cell proliferation and inducing apoptosis of neoplastic cells. Accomplishing the following aims will help determine the feasibility of developing inhibitors that block arachidonate-phospholipid remodeling as therapeutic agents that specifically inhibit the growth of neoplastic cells. Aim 1 will determine the relationship between intracellular levels of AA and the capacity of neoplastic cells to move through cell cycle check points. Experiments will be performed using hydroxyurea synchronized MDA-231 cells to determine whether the addition of poly-unsaturated fatty acids (PUFAs) and different classes of inhibitors that similarly induce intracellular AA accumulation can block cell cycle progression. Their ability to halt cell cycle progression at the same point within the cell cycle will be examined using DNA staining and flow cytometry analysis. Subsequent experiments will use flow cytometry and GC/MS to determine whether the accumulation of AA precedes the cell cycle block. Final experiments will utilize time lapse-videography to examine the capacity of several inhibitors that induce AA accumulation to cause early and late events of apoptosis. Aim 2 will elucidate the molecular mechanism by which the accumulation of AA leads to cell cycle arrest. Specifically, initial studies will determine whether increasing free AA within neoplastic cells produces novel oxidative products capable of influencing the proliferative or apoptotic status of neoplastic cells. Products will be isolated by established HPLC separation techniques and identified by GC/MS or HPLC/MS/MS. Another set of experiments will attempt to identify very potent anti-proliferative products produced by non-enzymatic oxidation of PUFAs, including auto-oxidation, copper ion and peroxide catalyzed reactions. Products will be isolated by HPLC methods and the MTT assay (tetrazolium ring is cleaved in active mitochondria, and thus living cells) will be used with MDA-cells to identify growth. Another set of experiments will examine whether attenuating environmental oxidative stress will influence the sensitivity of neoplastic cells to PUFAs or acylation and cyclooxygenase inhibitors.